三种隐孢子虫卵囊改良抗酸染色的形态学观察

目的观察3种隐孢子虫卵囊改良抗酸染色的形态特点。方法细胞图象分析系统观察改良抗酸染色后的牛隐孢子虫(C.bovis)、安氏隐孢子虫(C.andersoni)、隐孢子虫猪基因型Ⅱ(Cryptosporidium sp.pig genotypeⅡ)三种隐孢子虫卵囊的形态特点,测定卵囊长径和短径,计算卵囊指数。结果卵囊呈玫瑰红色,圆形或卵圆形,囊壁不着色,囊内子孢子的排列不规则且多形态,可见暗黑色颗粒状残留物。三种隐孢子虫卵囊中安氏隐孢子虫卵囊明显较其他两种卵囊大,但油镜下无法区分三种隐孢子虫卵囊形态。标本中常存在有大小不等、染色均匀、无内部结构的非特异性抗酸颗粒。结论改良抗酸染色法是较好的临床隐孢子虫卵囊检测方法,但需要与非特异性抗酸颗粒鉴别。     

应用巢式PCR方法诊断微小隐孢子虫感染的实验研究

目的应用巢式PCR扩增方法诊断微小隐孢子虫感染。方法用昆明种小鼠通过免疫抑制方法建立动物模型，通过不连续蔗糖密度梯度离心法分离、纯化隐孢子虫卵囊。抽提DNA后，用巢式PCR扩增隐孢子虫的18S核糖体DNA，电泳、割胶回收后测序。结果成功建立隐孢子虫的小鼠免疫抑制动物模型，纯化后的隐孢子虫卵囊形状均一，呈圆形或椭圆形，大小在3-6um左右。经巢式PCR扩增，在840bp左右有一条特异性条带。通过测序和生物信息学分析，证实该基因序列与微小隐孢子虫18S具有高度同源性。结论巢式PCR方法扩增18S基因是诊断微小隐孢子虫感染的敏感方法。     

从少量人源粪便中分离隐孢子虫用于全基因组测序的研究

目的 直接从少量人源粪便样本中分离、纯化隐孢子虫卵囊,提取并扩增DNA,使其浓度和纯度符合全基因组测序（WGS）要求。方法 2013年12月-2014年10月收集来自美国佛罗里达州、爱达荷州和缅因州11份少量人源隐孢子虫粪便样本,每份200 mg,使用免疫磁珠分离（IMS）进行纯化卵囊;2014年8-12月收集来自美国农业部52份牛源隐孢子虫粪便样本作为对照,每份2 000 mg,采用饱和蔗糖、氯化铯密度梯度离心法和IMS等方法纯化卵囊。均使用QIAamp DNA Mini试剂盒提取基因组DNA,然后采用REPLI-g MIDI试剂盒进行全基因组扩增（WGA）;最后通过定量PCR(qPCR)对WGA产物进行评估。结果 11份人源隐孢子虫WGA产物的DNA浓度为68.00～257.80 ng/μL。qPCR对隐孢子虫小亚基rRNA基因（SSU rRNA）扩增显示,11个分离株的循环数阈值（Ct）范围为8.92～19.23, Ct值越小表明隐孢子虫基因组DNA浓度越高;其中有6个分离株Ct <15,54.54%的分离株满足WGS要求。52份牛源隐孢子虫WGA产物的DNA浓度为27.2...     

水体中隐孢子虫卵囊的浓集和纯化

隐孢子虫为肠道寄生性原虫,可引起人兽共患病,隐孢子虫病暴发的主要途径是介水传播.水体中隐孢子虫的检测方法大致分为病原学检测、免疫学检测和分子生物学检测.由于水环境中隐孢子虫的含量相对较低,并且经处理后含量更低,同时为了排除环境水样中的干扰,因此在检测之前一般都需要把卵囊从浓集后的浓缩物中分离与纯化出来.浓集方法主要包括微滤膜及滤筒过滤法、超滤法、连续流式离心法、膜溶解法及絮凝沉淀法；分离纯化技术主要有浮聚法、密度梯度离心法及免疫磁珠分离法.本文即对水体中隐孢子虫卵囊的浓集以及分离纯化技术进行综述.     

隐孢子虫感染和隐孢子虫肠炎

隐孢子虫(Cryptosporidium)是一种肠道寄生性原虫,以往认为仅能引起动物腹泻,近年来发现隐孢子虫是能感染人的动物病(Zoonosis)或人畜共患病的病原体。 重庆医科大学附一院传染科罗明仪、王其南等为了解隐孢子虫在本地区的感染情况,用粪便三步检查法(第一步用2%碘液染色,寻找可疑隐孢子虫卵囊,一份粪标本检查三次;第二步用改良的Zie-hl-Neelsen抗酸染色法进一步认定卵囊,仍为一粪三检;第三步用Sheather氏糖液飘浮法观察隐孢子虫卵囊结构,凡第二、三步同时阳性者结合临床资料,有大便次数增多及性状改变者,定为隐孢子虫肠炎;无大便次数增多者定为隐孢子虫感染)检测了免疫功能正常者和受抑制者的粪标本共250份,发     

隐孢子虫病的病原学诊断及病例对照研究

目的为了验证接触动物、营养不良与隐孢子虫病感染有显著的统计学联系,探讨隐孢子虫病的病原学诊断方法。方法根据随机化的原则,在湖南省湘南地区用改良抗酸染色法检测隐孢子虫卵囊,以卵囊阳性为病例,卵囊阴性者为对照。将同一粪便标本一式二份,分别用碘染色法和改良抗酸染色法进行对比测试。结果接触动物者感染隐孢子虫的aoR=374,aOR=3.14。营养不良者感染隐孢子虫的aOR=4.66,aoR=4.35。碘染色法检出卵囊41例,阳性率1．9％,改良抗染色法检出卵囊41例,阳性率1.9％,符合率为100％。结论动物是人感染隐孢子虫的最重要传染源,接触动物是隐孢子虫感染的最重要的危险因素。营养不良所致的继发性细胞免疫功能缺陷及胃肠道形态学的功能变化,是造成易感染隐孢子虫病的危险因素。碘染色法简便,可靠,是检测粪便中CSO较理想的方法。     

隐孢子虫PCR检测体系的建立及其应用

目的建立快速检测隐孢子虫卵囊的PCR检测体系,以期在人群和食品及其饮用水腹泻原虫监测中推广应用。方法以隐孢子虫小亚基SSU r RNA和卵囊壁蛋白(COWP)为靶基因,设计巢式PCR和荧光PCR的引物和探针;以分离纯化后的微小隐孢子虫卵囊基因组DNA为模板进行扩增,分别进行两种检测方法的敏感性和特异性试验;再用某水牛养殖场采集的58份牛粪样考核检测体系的应用价值。结果设计的巢式PCR引物和荧光PCR引物探针特异性都很高,巢式PCR对隐孢子虫DNA的检测最低OD阈值为2 pg隐孢子虫DNA;实时荧光PCR显示最低检测阈值为200 fg隐孢子虫DNA,灵敏性都比较高,实时荧光PCR的灵敏性比巢式PCR高1个数量级(10倍);58份检测样本中,有一份为阳性样本,PCR产物经序列分析显示为微小隐孢子虫。结论巢式PCR和实时荧光PCR检测隐孢子虫的特异性和灵敏性均高,操作简便,能为隐孢子虫感染的诊治和预防提供有效的技术手段和方法依据。奶牛感染人兽共患微小隐孢子虫基因亚型,具有人兽共患传播的可能性。     

青岛地区哺乳动物隐孢子虫感染调查

目的 了解青岛地区哺乳动物隐孢子虫感染状况及最佳检测方法.方法 收集青岛地区某些县(市)的猪、狗、猫及青岛大学医学院寄生虫学教研室实验用小鼠、大鼠、兔粪便388份,分别应用改良抗酸染色、金胺-酚染色、金胺-酚串联改良抗酸染色法检测隐孢子虫,并在生理盐水、饱和盐水及碘液中对隐孢子虫卵囊进行形态学观察.用腹泻儿童粪便中分离隐孢子虫卵囊感染2只幼犬,对其进行病原学和病理学观察.结果 哺乳动物隐孢子虫检出率为15.98%(62/388),其中家养动物猪、狗和猫隐孢子虫检出率分别为4.71%(8/170)、22.22%(10/45)和0(0/15),实验动物小鼠、大鼠和兔隐孢子虫检出率分别为31.03%(9/29)、40.00%(24/60)和15.94%(11/69).实验动物隐孢子虫检出率(27.85%)明显高于家养动物(7.86%)(χ2=27.965,P<0.01).人隐孢子虫卵囊感染幼犬后产生明显腹泻症状,粪便中查到隐孢子虫卵囊,大小形态与人株相似,肠壁病变明显.结论 在青岛地区哺乳动物中查出隐孢子虫感染,金胺-酚串联改良抗酸染色法效果最好.对甲醛液保存时间较长的粪便标本,金胺-酚染色法染色后,用体积分数0.03盐酸-乙醇脱色5 min,再用改良抗酸法染色,可提高卵囊检出率.隐孢子虫人株对狗无宿主特异性,并且具有明显致病性.     

铜山县周棚村人群隐孢子虫感染调查

230份农村儿童成人粪便，采用改良抗酸染色法进行粪便涂片染色，发现隐孢子虫卵囊阳性者12例，感染率为5．22％，其中11例为儿童，平均年龄19．6个月。最大者则3岁，成人1例为女性，其子及侄女均为感染隐孢子虫。     

江苏省城乡儿童隐孢子虫感染

江苏省徐州市区和农村6个点隐孢子虫感染调查,≤15岁儿童5089例,隐孢子虫卵囊阳性89例,各点均有发现,平均感染率为1.75％(0.7～5.06％)。<4岁婴幼儿感染率明率高于4～15岁儿童(P<0.01)。部分病例血常规和免疫学检测,半数以上有贫血及细胞免疫功能低下。腹泻为本病主要临床表现,本组64％(57/89)出现腹泻,但亦发现较多的无症状带虫者。病例家人和畜粪便亦检获到隐孢子虫卵囊。     

食品和水源微小隐孢子虫18S rRNA鉴定方法研究

目的了解生蔬食品及相关水源微小隐孢子虫污染情况,并研究其卵囊分离及18S rRNA鉴定的方法. 方法采用分步离心富集法从78份生蔬食品和相关水源中分离出微小隐孢子虫卵囊并提取DNA模板,利用微小隐孢子虫18S rRNA 的基因序列设计特异性引物（446bp）进行多聚酶链反应扩增,扩增产物经回收克隆后进行PCR鉴定、酶切鉴定及序列同源性分析. 结果从78 份样品中检出4份特异性条带样品.限性样品的重组克隆经PCR鉴定可重现446bp的特异性条带,其酶切产物亦与目的基因PCR产物位置相同.重组菌液的测序结果与Geabank数据库中微小隐孢子虫18S rRNA基因序列的同源性为99%. 结论建立了一种监测生蔬食品及相关水源中微小隐孢子虫污染的基因检测方法.     

Cryptosporidiosis in an urban community

Over three months Cryptosporidium oocysts were identified in faecal samples from 43 (5%) of 867 patients presenting to their general practitioners with gastrointestinal symptoms. Cryptosporidium was the second most common enteric pathogen identified. Of the 867 patients, 329 were children aged under 5, of whom 24 (7%) excreted Cryptosporidium. A characteristic clinical presentation of infection with Cryptosporidium was recognised--namely, mild gastroenteritis with four to six watery, mucoid, and offensive motions a day, which lasted for one to two weeks. The source of infection was not identified, but direct contact with farm animals was not a feature and no association with a common water supply could be established.     

蒸馏水与稀释碘伏对结肠癌细胞SW480体外增殖的比较研究

目的研究蒸馏水与稀释碘伏对人结肠癌细胞SW480体外增殖的抑制作用。方法体外培养人结肠癌细胞株SW480,分别用蒸馏水与不同浓度稀释碘伏（0.05%,0.025%,0.01%）处理,MTT（噻唑蓝）法检测蒸馏水与稀释碘伏对SW480细胞的生长影响作用,并进行分析评价。结果 0.01%碘伏处理细胞1 min即可表现出明显的细胞毒性作用,效果远远高于单纯使用蒸馏水的杀伤作用。结论稀释碘伏在体外对人结肠癌细胞SW480的杀伤作用明显高于单纯使用蒸馏水,可望为临床手术中控制腹腔结肠癌细胞种植转移提供临床依据。     

Cryptosporidiosis and childhood diarrhoea in a rural community in Kenya

Cryptosporidiosis has come to recent prominence as a cause of diarrhoea throughout the world in both adults and children, producing either an acute self-limiting diarrhoea or a protracted chronic diarrhoea which can be fatal in immunosuppressed patients. This study was therefore conducted to assess the prevalence of the infection among children less than five years of age in a rural community in Kiambu District, Kenya. 1420 diarrhoea specimens from children less than five years which were processed for bacteriology and parasitology were also examined for Cryptosporidium oocysts, using the modified Ziehl-Neelsen (ZN) acid-fast stain, 3.8% of all the diarrhoea samples were positive for oocysts. 320 non-diarrhoeic stools from children in the same age group were also examined and were all negative for Cryptosporidium oocysts. The results of this study would imply that infection with Cryptosporidium is associated with acute childhood diarrhoea in Kenya.     

Cryptosporidium infections: a laboratory survey.

Between Oct. 1, 1983, and June 30, 1985, Cryptosporidium oocysts were identified in stool specimens from 74 patients who presented with gastrointestinal symptoms to their physicians. Questionnaires prepared to determine travel history, symptoms, duration of illness and epidemiologic characteristics of the infection were completed for 67 (90%) of the patients by their physicians; the information on the other 7 patients was obtained from the requisitions accompanying the specimens. Of the 67, 35 (52%) had recently been to Mexico. The infection was likely transmitted through contaminated water, food and, possibly, milk. The infections in patients who had not travelled were thought to be due to contact with infected pets or farm animals or with infected children attending daycare centres. Diarrhea, vomiting, fever and nausea usually lasted for 1 to 2 weeks, except in those with immune deficiency, in whom the symptoms persisted for up to 6 months. The condition was diagnosed by identification of oocysts in stool specimens that underwent formalin-ether sedimentation and modified cold Kinyoun staining.     

水体中隐孢子虫分子生物学检测方法进展

近年来,世界范围内水源性隐孢子虫病不断爆发。隐孢子虫卵囊壁对外界环境具有较强抵抗力,感染人的剂量较低,免疫力低下人群感染后症状较重,而临床上又无特效治疗药物。因此,及时检出水体中隐孢子虫卵囊的含量、种类及活力成为控制其大流行的首要任务。目前国际上推荐的隐孢子虫检测方法为美国EPA的1622或1623方法,但该方法造价高、过程繁琐且无法判断卵囊的种类及活力。各种PCR方法、LAMP方法、NASBA方法和核酸杂交等分子生物学方法则普遍具有速度快、灵敏度高、特异性好、费用低等特点,成为检测水体中隐孢子虫的重要手段和传统方法的有益补充。特别是分子生物学方法在卵囊类型判定和卵囊的活性有无的鉴别上普遍具有优势,因此还可以应用于隐孢子虫感染源的追踪溯源和污染环境的危害评估。其中,LAMP技术因其具有快速、特异性强、灵敏度高、成本低等优点,最具应用前景。     

The survey of Cryptosporidium infection among young children in kindergartens in Anhui province

Objective：To study the Cryptosporidium infection among young children in Anhui province, and to explore the best way to diagnose the disease. Methods： Stool specimens of 1 204 children were collected; oocysts of Cryptosporidium were identified with auramine O- modified staining, acid-fast staining, safranine T and methylene blue staining, and auramine O-modified acid-fast staining. Results： The detectable rate of Cryptosporidium in four stainings were respectively 2.46%, 1.50%, 1.98% and 3.46%, and there was a higher significant difference in the rate between auramine O-modified acid-fast staining and the others（P 〈 0.005）. The detectable rate was significantly lower in urban children（2.14%, 15/684） than in rural ones（5,19%, 27/520）. Boys and girls had similar detectable rate （1.99%, 24/1 204 vs. 1.50%, 18/1 204）. Cryptosporidium infection was usually subclinical, and its major clinical features included benign diarrhea, mild abdominal pain and nausea. Conclusion：Cryptosporidium infection was relatively common in kindergartens and a higher infection rate was found in rural children. As the majority of the Cryptosporidium infections were subclinical, diagnosis is important although difficult.     

荧光分光光度法测大黄中总蒽醌的含量

目的建立一种快速、简单、灵敏度高，测定大黄中总蒽醌的含量的荧光分光光度法。方法于10.0 ml比色管中加入一定量的1，8-二羟基蒽醌标准溶液，pH值为3.60的NaAc-HAc缓冲溶液2.0 ml，有机溶剂乙腈2.0ml，最后定容至10.0 ml；同时做空白对照，在最大激发波长为440 nm，最大发射波长为521 nm处测定一系列溶液的荧光强度。结果荧光强度与蒽醌的含量在0.0091-15.0μg/ml范围内呈良好的线性关系。用于大黄中总蒽醌的含量测定，结果令人满意。结论荧光分光光度法成功用于大黄中总蒽醌的含量测定。